Saccharomyces cerevisiae Final Risk Assessment

ATTACHMENT I--FINAL RISK ASSESSMENT OF

SACCHAROMYCESCEREVISIAE

(February 1997)

I. INTRODUCTION

Saccharomycescerevisiae has an extensive history of use
in the area of food processing. Also known as Baker's Yeast or Brewer's
Yeast, this organism has been used for centuries as leavening for bread
and as a fermenter of alcoholic beverages. With a prolonged history of
industrial applications, this yeast has been either the subject of or
model for various studies in the principles of microbiology. Jacob Henle
based his theories of disease transmission on studies of strains of Brewer's
Yeast. Currently, S. cerevisiae is the subject of a major
international effort to characterize a eucaryotic genome (Anderson, 1992).

History of Commercial Use and Products Subject to TSCA Jurisdiction

Saccharomycescerevisiae, in addition to its use in food
processing, is widely used for the production of macromolecular cellular
components such as lipids, proteins including enzymes, and vitamins (Bigelis,
1985; Stewart and Russell, 1985).

The Food and Drug Administration rates Brewer's Yeast extract as Generally
Recognized as Safe (FDA, 1986). Furthermore, the National Institutes of
Health in its Guidelines for Research Involving Recombinant DNA Molecules
(DHHS, 1986) considers S. cerevisiae a safe organism. Most
experiments involving S. cerevisiae have been exempted from
the NIH Guidelines based on an analysis of safety (see Appendix C-II of
the NIH Guidelines). While alcoholic beverages, vitamins, and bread leavening
are covered under the Federal Food, Drug and Cosmetic Act, the production
of enzymes and other macromolecules may be subject to TSCA regulation.
The abundance of information on S. cerevisiae, derived from
its role in industry, has positioned it as a primary model for genetic
studies and, by extension, as a strong candidate for genetic manipulation
for TSCA applications (Dynamac, 1990).

II. IDENTIFICATION AND CLASSIFICATION

A. Taxonomy and Characterization

Saccharomycescerevisiae is a yeast. The organism can
exist either as a singlecelled organism or as pseudomycelia. The cells
reproduce by multilateral budding. It produces from one to four ellipsoidal,
smoothwalled ascospores. S. cerevisiae can be differentiated
from other yeasts based on growth characteristics and physiological traits:
principally the ability to ferment individual sugars. Clinical identification
of yeast is conducted using commercially available diagnostic kits which
classify the organism through analysis of the ability of the yeast to
utilize distinct carbohydrates as sole sources of carbon (Buesching et
al., 1979; Rosini et al., 1982). More recently, developments in systematics
have led to the design of sophisticated techniques for classification,
including gasliquid chromatography of lysed whole cells (Brondz and Olsen,
1979).

As a result of the application of newer techniques arising from innovative
approaches, the taxonomy of Saccharomyces is subject to greater
scrutiny. The initial classification was based principally on morphological
characteristics with specific physiological and biochemical traits used
to differentiate between isolates with similar morphological traits. Using
these criteria, there are as many as 18 species listed in the literature.
In addition, what had been classified as one large heterogeneous species,
S. cerevisiae, may, in the future, be divided into four
distinct species based on DNA homology studies. The four species are S.
cerevisiae, S. bayanus (also known as S. uvarum),
S. pasteurianus (also known as S. carlsbergensis),
and S. paradoxus. All four represent industrially important
species. None of these organisms or other closely related species has
been associated with pathogenicity toward humans or has been shown to
have adverse effects on the environment.

Any assessment of Saccharomyces must take into consideration
the malleability of the current classification. For this assessment of
S. cerevisiae the reviews of the organism are based on the
classification proposed by Van der Walt (1971).

B. Related Species of Concern

None of the above strains or other closely related species has been
associated with pathogenicity toward humans or has been shown to have
adverse effects on the environment.

III. HAZARD ASSESSMENT

A. Human Health Hazards

1. Colonization and Pathogenicity

S. cerevisiae is a commonly used industrial microorganism
and is ubiquitous in nature, being present on fruits and vegetables. Industrial
workers and the general public come into contact with S. cerevisiae
on a daily basis through bothinhalation and ingestion (see section IV).
Saccharomyces spp. are frequently recovered from the stools and
throats of normally healthy individuals. This indicates that humans are
in constant contact with these yeasts.

There are individuals who may ingest large quantities of S. cerevisiae
every day, for example, people who take the yeast as part of a "health
food" regimen. Therefore, studies were conducted to ascertain whether
the ingestion of large numbers of these yeasts might result in either
colonization, or colonization and secondary spread to other organs of
the body. It was found that the installation of very large numbers of
S. cerevisiae into the colons of animals would result in
both colonization and passage of the yeasts to draining lymph nodes. It
required up to 1010S. cerevisiae in a single
oral treatment to rats to achieve a detectable passage from the intestine
to the lymph nodes (Wolochow et al., 1961). The concentrations of S.
cerevisiae required were well beyond those that would be encountered
through normal human daily exposure.

S. cerevisiae is not considered a pathogenic microorganism,
but has been reported rarely as a cause of opportunistic infections. Eng
et al. (1984) described five cases of such infections and reviewed the
literature on eight other S. cerevisiae infections (also
briefly reviewed by Walsh and Pizzo, 1988). All of the patients in the
cases had underlying disease. Some of them had also received antibiotic
therapy, thereby suppressing normal bacterial flora and allowing mycotic
organisms to become established.

A low concern for the pathogenicity of S. cerevisiae is
also illustrated by a series of surveys conducted at hospitals over the
last several years. S. cerevisiae accounted for less than
1% of all yeast infections isolated at a cancer hospital and in most of
the cases the organism was isolated from the respiratory system (Kiehn
et al., 1980). At YaleNew Haven Hospital over the past five years, there
have been 50 isolates of S. cerevisiae recovered from patients;
however, most of the isolates were considered contaminants (Dynamac, 1991).

2. Toxin Production

There have been no reports of isolates of S. cerevisiae
that produce toxins against either humans or animals. However, S.
cerevisiae has been shown to produce toxins against other yeasts.
These toxins, termed "killer toxins", are proteins or glycoproteins
produced by a range of yeasts. The yeasts have been genetically modified
to alter activity and are used in industrial settings as a means of controlling
contamination of fermentation systems by other yeasts (Sid et al., 1988).

3. Measure of the Degree of Virulence

A number of individual virulence factors have been identified as being
associated with the ability of yeasts to cause disease. The principal
virulence factors associated with yeasts appear to be phospholipase A
and lysophospholipase. It is believed that these enzymes enhance the ability
of the yeast to adhere to the cellwall surface and result in colonization
as a first step in the infectious process. Nonpathogenic yeast had considerably
lower phospholipase activities. Of a wide range of fungi assayed for phospholipase
production, S. cerevisiae was found to have the lowest level
of activity (BarrettBee et al., 1985). Therefore, based on the phospholipase
virulence factor S. cerevisiae is considered a nonpathogenic
yeast.

A second factor associated with virulence in yeast is the ability of
a fungus to impair the host's immune capabilities. The cell walls of most
fungi have the capacity to impede the immune response of the host. In
a study to determine the overall pathogenicity of a number of yeasts used
in industrial processes, animals exposed to both high levels of S.
cerevisiae and cortisone demonstrated a greater ability of the
fungus to colonize compared with those animals treated with only the yeast.
However, the animals suffered no illeffects from exposure to S.
cerevisiae (Holzschu et al., 1979). Therefore, this study suggests
that even with the addition of high levels of an immunosuppressant agent,
S. cerevisiae appears to be nonpathogenic.

4. Ability to Transfer Virulence Factor Genes

S. cerevisiae does not carry virulence factors to humans
or animals. However, the species does carry linear, doublestranded plasmids
which can be transmitted to other Saccharomyces. These plasmids
carry genes that encode the "killer toxins" discussed above
can be transferred from one Saccharomyces to another. Therefore,
gene constructs involving the incorporation of traits using these linear
plasmids should be considered to be nonstable.

5. Summary

In conclusion, S. cerevisiae is a organism which has an
extensive history of safe use. Despite considerable use of the organism
in research and the presence of S. cerevisiae in food, there
are limited reports in the literature of its pathogenicity to humans or
animals, and only in those cases where the human had a debilitating condition.
Factors associated with the virulence of yeasts (i.e., phospholipases)
indicate that this organism is nonpathogenic. The organism has not been
shown to produce toxins to humans.

B. Environmental Hazards

S. cerevisiae is ubiquitous in nature. It has been recovered
from a variety of sites under varying ecological conditions. The organism
is used in a variety of industrial scenarios. S. cerevisiae
is commonly recovered from a variety of fresh fruits and vegetables, generally
those fruits with high levels of fermentable sugars. However, it is not
listed as the causative agent of food spoilage for fruits and vegetables
(Phaff et al., 1966). The only adverse effect to the environment noted
in the literature is the presence of the "killer toxins" which
is active against other strains of Saccharomyces.

IV. EXPOSURE ASSESSMENT

A. Worker Exposure

S. cerevisiae is considered a Class 1 Containment Agent
under the National Institute of Health (NIH) Guidelines for Recombinant
DNA Molecules (U.S. Department of Health and Human Services, 1986).

No data were available for assessing the release and survival specifically
for fermentation facilities using S. cerevisiae. Therefore,
the potential worker exposures and routine releases to the environment
from large-scale, conventional fermentation processes were estimated on
information available from eight premanufacture notices submitted to EPA
under TSCA Section 5 and from published information collected from non-engineered
microorganisms (Reilly, 1991). These values are based on reasonable worst-case
scenarios and typical ranges or values are given for comparison.

During fermentation processes, worker exposure is possible during laboratory
pipetting, inoculation, sampling, harvesting, extraction, processing and
decontamination procedures. A typical site employs less than 10 workers/shift
and operates 24 hours/day throughout the year. NIOSH has conducted walk-through
surveys of several fermentation facilities in the enzyme industry and
monitored for microbial air contamination. These particular facilities
were not using recombinant microorganisms, but the processes were considered
typical of fermentation process technology. Area samples were taken in
locations where the potential for worker exposure was considered to be
potentially greatest, i.e., near the fermentor, the seed fermentor, sampling
ports, and separation processes (either filter press or rotary drum filter).
The workers with the highest potential average exposures at the three
facilities visited were those involved in air sampling. Area samples near
the sampling port revealed average airborne concentrations ranging from
350 to 648 cfu/m3. Typically, the Chemical Engineering Branch
would not use areamonitoring data to estimate occupational exposure levels
since the correlation between area concentrations and worker exposure
is highly uncertain. Personal sampling data are not available at the present
time. Thus, area sampling data have been the only means of assessing exposures
for previous PMN biotechnology submissions. Assuming that 20 samples per
day are drawn and that each sample takes up to 5 minutes to collect, the
duration of exposure for a single worker will be about 1.5 hours/day.
Assuming that the concentration of microorganisms in the worker's breathing
zone is equivalent to the levels found in the area sampling, the worst-case
daily inhalation exposure is estimated to range up to 650 to 1200 cfu/day.
The uncertainty associated with this estimated exposure value is not known
(Reilly, 1991).

B. Environmental and General Exposure

1. Fate of the Organism

S. cerevisiae is a normal inhabitant of soils and is widespread
in nature. S. cerevisiae is able to take up a wide variety
of sugars and amino acids. These traits enhance the organism's ability
for long term survival. S. cerevisiae can be isolated from
fruits and grains and other materials with a high concentration of carbohydrates
(LaVeck, 1991).

2. Releases

Estimates of the number of S. cerevisiae organisms released
during production are tabulated in Table 1 (Reilly, 1991). The uncontrolled/untreated
scenario assumes no control features for the fermentor offgases, and no
inactivation of the fermentation broth for the liquid and solid waste
releases. The containment criteria required for the full exemption scenario
assume the use of features or equipment that minimize the number of viable
cells in the fermentor off-gases. They also assume inactivation procedures
resulting in a validated 6log reduction of the number of viable microorganisms
in the liquid and solid wastes relative to the maximum cell density of
the fermentation broth.

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TABLE 1. Estimated Number of Viable Saccharomycescerevisiae

Organisms Released During Production

Uncontrolled/ Full

Release Media Untreated Exemption Release

(cfu/day) (cfu/day) (days/year)

_________________________________________________________________

Air Vents 2x108 - 1x1011 <2x108 - 1x1011 350

Rotary Drum Filter 250 250 350

Surface Water 7x1012 7x106 90

Soil/Landfill 7x1014 7x108 90

_________________________________________________________________

Source: Reilly, 1991

These are "worstcase" estimates which assume that the maximum
cell density in the fermentation broth for fungi is 107 cfu/ml,
with a fermentor size of 70,000 liters, and the separation efficiency
for the rotary drum filter is 99 percent.

3. Air

Specific data which indicate the survivability of S. cerevisiae
in the atmosphere after release are currently unavailable. Survival of
vegetative cells during aerosolization is typically limited due to stresses
such as shear forces, desiccation, temperature, and UV light exposure.
As with naturally-occurring strains, human exposure may occur via inhalation
as the organisms are dispersed in the atmosphere attached to dust particles,
or lofted through mechanical or air disturbance.

Air releases from fermentor offgas could potentially result in nonoccupational
inhalation exposures due to point source releases. To estimate exposures
from this source, the sector averaging form of the Gaussian algorithm
described in Turner (1970) was used. For purposes of this assessment,
a release height of 3 meters and downward contact at a distance of 100
meters were assumed. Assuming that there is no removal of organisms by
controls/equipment for offgases, potential human inhalation dose rates
are estimated to range from 3.0 x 103 to 1.5 x 106
cfu/year for the uncontrolled/untreated scenario and less than that for
systems with full exemptions. It should be noted that these estimates
represent hypothetical exposures under reasonable worst case conditions
(Versar, 1992).

4. Water

The concentrations of S. cerevisiae in surface water were
estimated using stream flow values for water bodies receivingprocess wastewater
discharges from facilities within SIC Code 283 (drugs, medicinal chemicals,
and pharmaceuticals). The surface water release data (cfu/day) tabulated
in Table 1 were divided by the stream flow values to yield a surface water
concentration of the organism (cfu/l). The stream flow values for SIC
Code 283 were based on discharger location data retrieved from the Industrial
Facilities Dischargers (IFD) database on December 5, 1991, and surface
water flow data retrieved from the RXGAGE database. Flow values were obtained
for water bodies receiving wastewater discharges from 154 indirect (facilities
that send their waste to a POTW) and direct dischargers facilities that
have a NPDES permit to discharge to surface water). Tenth percentile values
indicate flows for smaller rivers within this distribution of 154 receiving
water flows and 50th percentile values indicate flows for more average
rivers. The flow value expressed as 7Q10 is the lowest flow observed over
seven consecutive days during a 10year period. The use of this methodology
to estimate concentrations of S. cerevisiae in surface water
assumes that all of the discharged organisms survive wastewater treatment
and that growth is not enhanced by any component of the treatment process.
Estimated concentrations of S. cerevisiae in surface water
for the uncontrolled/untreated and the full exemption scenarios are tabulated
in Table 2 (Versar, 1992).

TABLE 2. S. cerevisiae Concentrations in Surface Water

Receiving

Flow Stream Flow Organisms

(MLD*) (cfu/l)

_________________________________________

Mean 7Q10 Mean 7Q10

_________________________________________________________________

Uncontrolled/Untreated

10th Percentile 156 5.60 4.5x104 1.25x106

50th Percentile 768 68.13 9.11x103 1.03x105

Full Exemption

10th Percentile 156 5.60 4.5x10-2 1.25x1000

50th Percentile 768 68.13 9.11x10-3 1.03x10-1

_________________________________________________________________

*MLD = million liters per day

Source: Versar, 1992

5. Soil

Since soil is a natural habitat for S. cerevisiae, it
would be expected to survive well in soil. These releases could result
in human and environmental exposure (Versar, 1992). It iscurrently estimated
that over one million tons of naturally-occurring yeast are produced annually
during brewing and distilling practices (LaVeck, 1991).

6. Summary

Although direct monitoring data are unavailable, worst case estimates
do not suggest high levels of exposure of S. cerevisiae
to either workers or the public resulting from normal fermentation operations.

V. INTEGRATED RISK ASSESSMENT

A. Discussion

There is an extensive history of use of and exposure to S. cerevisiae
with a very limited record of adverse effects to the environment or human
health. Yeast has been used for centuries as a leavening for bread and
fermenter of beer without records of virulence. S. cerevisiae
is currently classified as a class 1 containment organism under the NIH
Guidelines based largely on the extensive history of safe use.

Factors associated with the development of disease states in fungi have
been reviewed. Data suggests that only with the ingestion of high levels
of S. cerevisiae or with the use of immunosuppressants can
S. cerevisiae colonize in the body. Even under those conditions,
there were no noted adverse effects. In the few cases which S.
cerevisiae was found in association with a disease state, the host
was a debilitated individual, generally with an impaired immune system.
In other cases the organism was recovered from an immunologically privileged
site (i.e., respiratory tract). Many scientists believe that under appropriate
conditions any microorganism could serve as an opportunistic pathogen.
The cases noted in the above Human Health Assessment, where S.
cerevisiae was found in association with a disease state, appear
to be classic examples of opportunistic pathogenicity (see III.A.3).

The organism is not a plant or animal pathogen. Despite the fact that
S. cerevisiae is ubiquitous in nature, it has not been found
to be associated with disease conditions in plants or animals. The only
adverse environmental condition that was noted is the production of "killer
toxins" by some strains of the yeast. These toxins have a target
range that is limited to susceptible yeasts. The toxins, proteins and
glycoproteins, are not expected to have a broad environmental effect based
largely on the anticipated short persistence of the toxins in soil orwater
and by the limited target range. S. cerevisiae "killer
toxin" has been used industrially to provide a level of protection
against contamination by other yeasts in the fermentation beer.

The current taxonomy of Saccharomyces is under revision based
on the development of alternative criteria. However, this should not have
a major effect on the risk associated with closely related species. Saccharomyces,
as a genus, present low risk to human health or the environment. Criteria
used to differentiate between species are based on their ability to utilize
specific carbohydrates without relevance to pathogenicity. Nonetheless,
this risk assessment applies to those organisms that fall under the classical
definition of S. cerevisiae as described by van der Walt
(1971).

S. cerevisiae is a ubiquitous organism which, despite
its broad exposure, has very limited reported incidence of adverse effects.
The extensive history of use, the diversity of products currently produced by the organism, and the attention given
this organism as a model for genetic studies collectively makes this organism
a prime candidate for full exemption. The increased knowledge derived
from the ongoing research should further enhance this organisms' biotechnological
uses.